Today there are many approaches to identifying chemical entities which have a desired effect on a pharmaceutical target and therefore potential as drugs. Common to all of these processes is the sequential use of multiple assays to identify a test compound's composite activity profile. This activity profile usually consists of information on four basic attributes: potency, activity, selectivity and specificity. Selectivity indicates the ability to distinguish among closely related members of a particular target family. Specificity is the ability to distinguish between unrelated targets. Only two types of assays are used to develop the activity profile of a potential drug: one, a binding assay to measure affinity (i.e. potency) of the compound; and a second, an activity assay, to measure the compounds effect (i.e. agonistic or antagonistic) on the target. Binding assays measure the formation of the complex between target (T) and ligand (L). Targets include receptors, enzymes or structural components. Ligands include signals such as hormones, neurotransmitters, growth factors or test compounds. Until recently, L was labelled in some fashion (L*) for identification and quantitation of the L:T complex. Recently, binding assays have been developed which use a tagged R (R*) to assess L affinity (see below). All these processes of labelling and R:L complex isolation and quantitation are known to those skilled in the art and have been reviewed.
In the process of searching for small organic molecules with appropriate potency, activity, selectivity and specificity for a particular pharmaceutical target, the order of testing is most often affinity, activity, selectivity and then specificity. In addition, some form of binding and/or activity assay, is interspersed with synthetic chemistry efforts at improving the compounds attributes. This generates an iterative cyclic discovery processes in which various assays and synthesis are repeated over until a compound possessing all of the desirable properties is obtained.
The present iterative process, although successful, is extremely time consuming and has a high probability of failure for several reasons. Although binding and activity assays have now been automated, screening takes significant time as it is done on individual entities within chemical files containing over 100,000 entities. In addition, the properties of potency, activity, specificity and selectivity are separable, such that the presence in a compound of any one property is not predictive of attaining another. For example, binding assays give no conclusive data on the activity (i.e., a compound with high affinity may be an antagonist), and activity assays do not predict selectivity or affinity. As a result, modifying a compound so as to change one of its attributes (i.e., agonist activity) without modifying another (i.e., target affinity or selectivity) is unpredictable and considerable time is added to the discovery program when high affinity compounds identified early in the discovery process turn out to have inappropriate activity or selectivity.
The relatively large number of biologically active small organic ligands having different general structures and which are capable of binding to a particular pharmaceutical target suggest that the binding surface of the target is not singularly unique. Furthermore, binding assays using an endogenous ligand or close analog thereof are inherently biased to compounds which bind to only a fraction of the available surface of the target. Even where the labelled ligand is not an endogenous one, this confinement means that the vast majority of active compounds identified by this process will be greatly restricted to the surface domain of the target which is used for interaction with the endogenous ligand.
This limitation is often viewed as desirable because the recognition domain for the endogenous ligand are those known via previous studies to have the ability to modify target activity. However, investigation of only one target area severely restricts the ability to identify useful ligands. As endogenous ligands in most instances are agonists peptides as in the case of opiate receptors, antagonist discovery can become a rare event. In addition, because endogenous binding domains often exhibit limited diversity among receptor members of a single target family, it becomes difficult for active compounds to discriminate among target family members. This often occurs when the endogenous signal for the family is a single entity and not a group of closely related entities. Acetylcholine (ACh) receptors are an example of a target family with only one signal entity. The catecholamine receptors are an example of a target family with a few but highly related endogenous catechol signals.
In many cases, target diversity is found in target domains other than the specific binding site of the endogenous ligand. Some of these domains may be associated with the target's other functions, i.e., signal transmission while others are quiescent domains not being used by any endogenous signals recognition or transmission. An example of a dilemma in discriminating among target family members is that found for the muscarinic receptor family (AChRm) where the binding domain for acetylcholine is used to monitor a test compound's potency, yet finding AChRm agonists which distinguish among the five ACHRm subtypes has proven illusive to date.
The task for drug discovery is to devise a screening approach which provides detectable ligands to be used to screen compounds which bind to the target and provide information regarding potency, activity, specificity and selectivity, as well as the three dimension (3D) conformation of compounds active at that particular site on the pharmaceutical target.
As part of any solution of these problems it is also necessary to establish binding assays which report the interaction of test compounds with allosteric modulatory sites on targets. An allosteric site is one which modifies the endogenous ligand binding site yet is discontinuous and non-overlapping with that site. Such target sites have important physiological and pharmaceutical consequences and have been reported. For example, the allosteric site on the Gaba A receptor binds benzodiazepines (BDZ) and thereby modulates the binding of the endogenous neurotransmitter Gaba. Occupation of the allosteric BDZ site, which can be done by chemicals from many unrelated structural groups, has a significant and recognized therapeutic influence on physiological processes including anxiety and sedation.
It is also known that active allosteric sites exist which are modulatory for endogenous ligand binding and have observable effects of their own on the target. Such an allosteric site is present on the Gaba receptor. [Garrett, Blume and Abel 1986; Garrett, Abel and Blume 1986].
Present screening techniques which monitor direct binding of test compounds to allosteric target sites are not routinely done because a) high affinity tagged ligands which bind to these sites are usually unavailable at the start of a discovery program; and b) the necessary monitoring of detectable endogenous ligand dissociation or bioassays are too time consuming in initial screening protocols. Without a simple, rapid and comprehensive way to observe all potential target sites, investigation of the surface of a pharmaceutical target for potential modulation remains limited to a small part of the target surface. New methods are necessary to survey the entire target surface in early screening for discovery leads.
Recently methods of identifying various entities which recognize target surfaces have been reported which do not depend upon the availability of tagged ligands with high affinity for the target. [Delvin, J. J., Panganiban, L. C., and Devlin, P. E., 1990]. These assays detect a compounds surface recognition activity directly via formation of an identifiable tagged target (T*):Ligand complex. In one version, test compound is coupled in identifiable compartments to a solid matrix of varied composition at concentrations which allow sufficient amounts of labelled target to bind and form stable ligand-labelled target complexes for subsequent detection via chemical, radioactive, or biological methods known to those skilled in the art. Subsequent isolation (or identification) of test compound from the compartments containing labelled target provide active chemical structures. In one such version where test compounds are free oligonucleotides, the oligonucleotides are isolated in complexes with the target, and are amplified and sequenced by PCR technology. [Delvin, J. J., Panganiban, L. C., and Devlin, P. E., 1990].
Phage display is a particularly sensitive method of presenting peptide test compounds to a target. Phage may be engineered to express the gene encoding the test peptide as a fusion protein with one of its surface proteins. Methods involving phage display are referred to in Winter et al. PCT application WO 92/20791; Huse, WO92/06204; and Ladner et al. WO90/02809.
Although these newer approaches have now been incorporated into random drug screening protocols, they do not resolve the following problems: the assays of the critical attributes of potency, activity, selectivity and specificity are still unconnected; active target surfaces including the endogenous ligand site and allosteric sites have not been identified; and 3D information on conformation of the active agent is not provided. More importantly, most of the agents available for screening, i.e., peptides, nucleotides, lipids, and carbohydrates which are available in large libraries, are not totally satisfying as discovery leads because none are expected to be orally active, or pass membrane barriers to get at intracellular or central nervous system targets. In addition, these classes of compounds are so flexible as to obscure their active 3D-configuration to such a degree as to prevent or severely limit their use as models for organic synthetic efforts. An improvement in screening would then encompass a resolution of these deficiencies so that these broad surface recognition libraries could attain their full usefulness.
In covering the prior art for high throughput binding screens for target modifiers, it also is necessary to review what is known of the endogenous ligand signals as well as their targets. Both shed significant light on additional problems and limitations encountered in the binding assays available today for discovery approaches.
Endogenous ligand signals are those ligands which directly modify target activity. The size of endogenous ligands varies greatly, ranging from 100 Daltons (e.g., as for glycine in its regulatory role as an excitatory amino acid neurotransmitter) to over 100 kD (e.g., as for some extracellularly active growth factors (GF) with a proportioned increase on surface area. The composition of endogenous ligand is equally varied including organics such as neurotransmitters; peptides e.g. somatostatin, LH, LHRH and TRH; proteins eg., growth factors; and lipids; carbohydrates; and inorganics such as ions.
For discovery purposes, common to all is the desire to replace the endogenous ligand with a small organic molecule. The problem of screening for replacements appears to be very different for most small endogenous ligands, i.e, neurotransmitters and neuropeptide modulators compared to large endogenous ligands i.e., hormones, growth and differentiation factors. Although small organic molecules have been found which can be active at targets for small endogenous ligands, few, if any have been found for the larger molecules such as proteins.
Corresponding to the diversity in endogenous ligands is the equally extensive diversity in target domains which are responsible for recognizing (i.e., binding) and responding to endogenous ligand signals. It is generally accepted that both signal and target have specific domains involved in forming the actual contact points found within the endogenous ligand:target complex (EnL:T). Recent data on crystallized growth hormone (GH) and its receptor complex provides detailed molecular information on the amino acids within the GH hormone ligand and its target GH receptor interactive domains.
Recent data on the crystal structure of GH and its receptor has shown a single GH molecule to contact the same set of amino acids in each of two identical GH receptor units complexed with one GH molecule. [Cunningham and Wells 1989; Cunningham et al. 1991; DeVos, et al. 1992]. Each of the receptor units therefore has only one target site which is the same on both units. Each receptor uses the same 7 amino acids to define the binding site which participate in GH binding and receptor dimerization necessary for activity. [Cunningham and Wells 1989; Cunningham et al. 1991; DeVos, et al. 1992].
Dimerization of at least two receptor subunits by monomeric or multimeric hormones is required for receptor activation for the majority of hormones studied to date, such as growth factors, including nerve growth factor (NGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), interleukins (IL2, 4 and 6), interferons and insulin. [DeFronzo, Bonadonna, and Ferrannini, 1992; Bamborough, Hedgecock and Richards 1994; Kishimoto, et al. 1994; Claesson-Welsh, 1995]. In some cases, the two units of the hormone, as well as receptor are not genetically related. In such cases one subunit provides high affinity hormone binding and the other intracellular signalling (e.g., tyrosine kinase activity). [Ullrich, et al., 1986; Kaplan, Martin-Zanco, and Patrada, 1991; Kaplan, et al. 1991; Klein, et al. 1991; Argetsinger, et al. 1993; Obermeier, et al. 1993; Weiss 1993]. In some cases, the lower affinity receptor when dimerized can be activated. [Ullrich and Schlessinger 1990; Stahl and Yancopoulos 1993; Claesson-Welsh 1995].
Among many hormones and hormone receptors, it is now apparent that an unexpected and unanticipated degree of structural homology exists with subgroups of these signals and receptors forming homologous families which sometimes follow along different genetic evolutionary lines. Other functional similarities may be brought about as a result of convergent evolution. In either case, the active 3D conformations of ligands and receptors appear to follow some general principals. However, for drug discovery, the principals gleaned from these studies have not yet been detailed enough to bypass crystallography of particular hormone/receptor complexes in order to gain sufficient specific information as to deduce the molecular shape of active small organic molecules.
Deciphering the elements necessary in a signal to activate a hormone/growth factor receptor has included (1) crystal formation and analysis at &lt;3 .ANG. of receptor and endogenous ligand complexes; (2) the influence on function (i.e, ligand binding and receptor activation) caused by molecular biological mutagenesis of single amino acids or short peptide deletion/replacement, or chimera formation of both the hormone and receptor units. In addition, monoclonal antibody binding to surface domains available when ligand and receptor are either uncomplexed or in the R:L complex, along with the ability of Fab2 versus Fab1 to activate or block receptor activation in vitro, in situ or in vivo has been studied.
The above studies when taken together, provide information concerning (1) the contact points between hormone and receptor; (2) the amount of energy of binding involved in these contact points; (3) amino acids outside of the receptor:ligand contact points essential for global receptor/ligand stability or dimer stability, or receptor signalling activity (i.e. tyrosine kinase, binding of other intracellular regulatory factors, internalization, uncoupling for effector system).
Critical for identifying small organic molecules which are active at hormone receptors are the data from the above indicating (1) number of units/active complex; (2) amino acids of the target specifically involved in the binding domain with the endogenous ligand; and 3) amino acids of the ligand specifically involved in binding and/or activating the target. Of all of the above information, clearly the rate limiting event today is obtaining sufficiently resolved crystallography data of hormone/receptor complexes. However, complexes of receptor and ligand are often difficult to identify and crystalize thus preventing one from obtaining the structural information. It is also recognized that the various molecular, biological, immunological studies, biochemical and pharmacological studies noted above, also take considerable time and effort. Accordingly, prior art approaches to identifying active small organic molecules are long and arduous with unpredictable results.
In the approach outlined above, it is important that both structural and biological data be obtained as each has its own limitations and artifacts. Also, contact points could reflect specific aspects of crystal formation which do not reflect the structure at the protein in situ, or the crystal may contain an inappropriate number of subunits. On the other hand, the biological data generates both false positives and negatives. Furthermore, if antibodies are used to probe the binding site of the target, not all receptor or ligand surfaces may be immunogenetic accessible to Fab2 or Fab1 antibody. Another problem is the difficulty studying allosteric sites which do not interact directly with the signal ligand.
Despite considerable effort, a major problem in drug discovery has been the identification of small organic molecules capable of activating peptide hormone/growth factor receptors. This is likely the result of the multivalent nature of endogenous ligands for these receptors and the requirement to dimerize or simultaneously activate multiple attachment sites on a single receptor (receptor subunits) for receptor activation. Even for receptors which are homodimers, such as GH receptor (GHR), a single small organic molecular monovalent attachment to the GHR site I is not sufficient to cause activation, nor displacement of growth hormone from its active divalent dimer receptor complex.
Failure to find single small organic molecules in conventional binding assays stems from the fact that the labelled hormone is bivalent, and its displacement from two receptor units by a single monovalent small organic molecule (i.e. compounds which attach to only one receptor target at a time) is thermodynamically unfavorable in the present day binding assay. Furthermore, in the large majority of cases the receptor for a given hormone is a heterodimer. Thus, for a given hormone/growth factor-receptor binding pair, there may exist at least two different binding sites on the target which may be due to the multimeric nature of the target or a target consisting of allosteric sites on a monomeric unit. In all of these cases, the endogenous ligand must therefore comprise at least a sufficient number of binding sites which are properly spaced to bind to the multiple sites on the target necessary for activation. Obviously, one would then require a multimeric or a multivalent small organic molecule for displacement of these hormones from their targets.
Given the complexity required of each small organic molecule to bind the receptor at the multiple sites necessary for activity, or to displace the endogenous ligand, one could expect that the occurrence of a single small organic molecule with two unrelated yet active binding domains would be equal to the chance of finding one multiplied by the chance of finding the other independently. As active small organic molecules are found by random robotic assays at a frequency of between 1/1000 to 1/10,000 on most screens for ligands requiring only one binding site on the ligand, and which have correspondingly a single binding site on the receptor, one would expect to screen an organic chemical libraries containing from 10.sup.6 to 10.sup.8 compounds in order to identify an active molecule. Such libraries exceed those which could be screened in some reasonable assay format and actually exceed most made by even the largest pharmaceutical companies.
Therefore, a different approach to screening for small organic molecules which can activate hormone receptors is needed.
A number of libraries now exist for screening such large numbers. Two have been noted already, the oligonucleotide and peptide library. Another such file contains natural products.
Classical chemical libraries consisting of synthetically derived small organic molecules are routinely available from commercial sources (e.g. Alldrich, and Kodak) and consist of upwards of a 1-200,000 entities. Recently a survey of the chemical entities within such libraries uncovered 100,000 or so chemical structures as being the cores upon which most of the individual entities were crafted. The average molecular weight of the entity within such files ranges between 200-400 Daltons which would account for no more than one such contact site per target.
Screening of small chemical compound libraries is limited only by their availability, which most often is &lt;100,000.
With the advent of molecular biology and gene cloning and sequencing, it has been discovered that most pharmaceutical targets are not unique entities unto themselves, but in fact belong to families of sometimes rather large size and close relatedness. Recognition of this fact has mandated a much more serious look at all of the members of the family to which the target under investigation belongs so as to identify lead compounds which can distinguish among its family members. If one used only binding assays as a primary screen for potency, activity, selectivity and specificity, one would require affinity labelled standards for each of the family members. Although this is potentially possible when the endogenous ligand signal are proteins due to their native affinity and ease of labelling, it is not presently feasible where small organics are the only known signals. This approach is also unsuitable for targets with unidentified signal ligands. Any discovery of how to include such widespread specificity testing into primary binding screen assays would greatly increase the probability of drug discovery success.